We are focusing our studies on proving or disproving the hypothesis that TRPC type cation channels are the mediators of store depletion activated calcium entry. In earlier studies we had discovered six of the seven TRPC channels, cloned full length cDNAs of four (TRPC1, TRPC2, TRPC3 and TRPC6) and shown them to be activated by maneuvers that stimulate the Gq-PLCb-IP3 mediated depletion of calcium stores. Focusing on TRPC3, and showed that peptides of the IP3 receptor that interact in vitro with TRPC3 segments (GST pull-down) affect store depletion activated calcium entry. But direct activation of TRPC3 upon thapsigargin stimulated store depletion independent of G protein-PLCbeta activation failed to show the classical capacitative calcium entry response. During the previous year we discovered that TRPC3 is subject to phosphorylation on several tyrosines located on its N-terminus. Of these, tyrosine 226 is critical for function. Since submitting the FY04 Progress report we confirmed that Y226 is phosphorylated by src. We now extended our studies: The closely related tyrosine kinases yes and fyn cannot substitute for src in phosphorylating TRPC3 Y226. TRPC3 is structurally closely related to TRPC6 and TRPC7 sharing 70-80% sequence identity from aa 26 through 426 and the property of being activated by DAG, absent in TRPC1, 4, 5 and 5. Surprisingly, neither TRPC6, nor TRPC7 share the dependence on a src-type tyrosine kinase for their activation, indicating that TRPC3 is unique in its absolute dependence in an N-terminal tyrosione for its activation. The new, N-terminally extended TRPC3 (TRPC3a) was shown to activated by store depletion, thus joining TRPC1, 2, 4 5 and 7 in having the capacity to respond to store depletion. Phenotyping the TRPC6 KO mouse, generated by us in 2000 at UCLA, revealed increased vascular smooth muscle contractility, which is caused by unchecked overexpression and activity of TRPC3. We have obtained germline transmission for TRPC3 and TRPC5 with floxed exons. in preparation for generating by breeding conditional KO mice, and are expecting to obtain the same for TRPC7 very soon. These will add to our previously generated classical knockouts of TRPC1 and TRPC6 and will serve to further investigate the physiological roles of the TRPC family of TRP channels.